Florian Wüst

A Third Phytoene Synthase Is Devoted to Abiotic Stress-Induced Abscisic Acid Formation in Rice and Defines Functional Diversification of Phytoene Synthase Genes

We here report on the characterization of a novel third phytoene synthase gene (PSY) in rice (Oryza sativa), OsPSY3, and on the differences among all three PSY genes with respect to the tissue-specific expression and regulation upon various environmental stimuli. The two already known PSYs are under phytochrome control and involved in carotenoid biosynthesis in photosynthetically active tissues and exhibit different expression patterns during chloroplast development. In contrast, OsPSY3 transcript levels are not affected by light and show almost no tissue-specific differences. Rather, OsPSY3 transcripts are up-regulated during increased abscisic acid (ABA) formation upon salt treatment and drought, especially in roots. The simultaneous induction of genes encoding 9-cis-epoxycarotenoid dioxygenases (NCEDs), involved in the initial steps of ABA biosynthesis, indicate that decreased xanthophyll levels are compensated by the induction of the third PSY gene. Furthermore, OsPSY3 and the OsNCEDs investigated were also induced by the application of ABA, indicating positive feedback regulation. The regulatory differences are mirrored by cis-acting elements in the corresponding promoter regions, with light-responsive elements for OsPSY1 and OsPSY2 and an ABA-response element as well as a coupling element for OsPSY3. The investigation of the gene structures and 5′ untranslated regions revealed that OsPSY1 represents a descendant of an ancient PSY gene present in the common ancestor of monocots and dicots. Since the genomic structures of OsPSY2 and OsPSY3 are comparable, we conclude that they originated from the most recent common ancestor, OsPSY1.

Carotenoid Crystal Formation in Arabidopsis and Carrot Roots Caused by Increased Phytoene Synthase Protein Levels

Background As the first pathway-specific enzyme in carotenoid biosynthesis, phytoene synthase (PSY) is a prime regulatory target. This includes a number of biotechnological approaches that have successfully increased the carotenoid content in agronomically relevant non-green plant tissues through tissue-specific PSY overexpression. We investigated the differential effects of constitutive AtPSY overexpression in green and non-green cells of transgenic Arabidopsis lines. This revealed striking similarities to the situation found in orange carrot roots with respect to carotenoid amounts and sequestration mechanism. Methology/Principal Findings In Arabidopsis seedlings, carotenoid content remained unaffected by increased AtPSY levels although the protein was almost quantitatively imported into plastids, as shown by western blot analyses. In contrast, non-photosynthetic calli and roots overexpressing AtPSY accumulated carotenoids 10 and 100-fold above the corresponding wild-type tissues and contained 1800 and 500 µg carotenoids per g dry weight, respectively. This increase coincided with a change of the pattern of accumulated carotenoids, as xanthophylls decreased relative to β-carotene and carotene intermediates accumulated. As shown by polarization microscopy, carotenoids were found deposited in crystals, similar to crystalline-type chromoplasts of non-green tissues present in several other taxa. In fact, orange-colored carrots showed a similar situation with increased PSY protein as well as carotenoid levels and accumulation patterns whereas wild white-rooted carrots were similar to Arabidopsis wild type roots in this respect. Initiation of carotenoid crystal formation by increased PSY protein amounts was further confirmed by overexpressing crtB, a bacterial PSY gene, in white carrots, resulting in increased carotenoid amounts deposited in crystals. Conclusions The sequestration of carotenoids into crystals can be driven by the functional overexpression of one biosynthetic enzyme in non-green plastids not requiring a chromoplast developmental program as this does not exist in Arabidopsis. Thus, PSY expression plays a major, rate-limiting role in the transition from white to orange-colored carrots.

Photoconversion and Nuclear Trafficking Cycles Determine Phytochrome A's Response Profile to Far-Red Light

Phytochrome A (phyA) is the only photoreceptor in plants, initiating responses in far-red light and, as such, essential for survival in canopy shade. Although the absorption and the ratio of active versus total phyA are maximal in red light, far-red light is the most efficient trigger of phyA-dependent responses. Using a joint experimental-theoretical approach, we unravel the mechanism underlying this shift of the phyA action peak from red to far-red light and show that it relies on specific molecular interactions rather than on intrinsic changes to phyAs spectral properties. According to our model, the dissociation rate of the phyA-FHY1/FHL nuclear import complex is a principle determinant of the phyA action peak. The findings suggest how higher plants acquired the ability to sense far-red light from an ancestral photoreceptor tuned to respond to red light.

Interaction with plant transcription factors can mediate nuclear import of phytochrome B

Phytochromes (phy) are red/far-red–absorbing photoreceptors that regulate the adaption of plant growth and development to changes in ambient light conditions. The nuclear transport of the phytochromes upon light activation is regarded as a key step in phytochrome signaling. Although nuclear import of phyA is regulated by the transport facilitators far red elongated hypocotyl 1 (FHY1) and fhy1-like, an intrinsic nuclear localization signal was proposed to be involved in the nuclear accumulation of phyB. We recently showed that nuclear import of phytochromes can be analyzed in a cell-free system consisting of isolated nuclei of the unicellular green algae Acetabularia acetabulum. We now show that this system is also versatile to elucidate the mechanism of the nuclear transport of phyB. We tested the nuclear transport characteristics of full-length phyB as well as N- and C-terminal phyB fragments in vitro and showed that the nuclear import of phyB can be facilitated by phytochrome-interacting factor 3 (PIF3). In vivo measurements of phyB nuclear accumulation in the absence of PIF1, -3, -4, and -5 indicate that these PIFs are the major transport facilitators during the first hours of deetiolation. Under prolonged irradiations additional factors might be responsible for phyB nuclear transport in the plant.

Characterization of phytoene synthases from cassava and their involvement in abiotic stress-mediated responses

Abiotic stress stimuli induce the increased synthesis of abscisic acid (ABA), which is generated through the cleavage of xanthophyll precursors. To cope with the increased xanthophyll demand, maize and rice contain a third stress-induced gene copy, coding for phytoene synthase (PSY), which catalyzes the first carotenoid-specific reaction in the pathway. To investigate whether this specific response extends beyond the Poaceae, cassava was analyzed, an important tropical crop known for its drought tolerance. We also found three PSY genes in cassava, one of which (MePSY3) forms a separate branch with the stress-specific Poaceae homologs. However, MePSY3 transcripts were virtually absent in all tissues investigated and did not change upon abiotic stress treatment. In contrast, the two remaining PSY genes contributed differentially to carotenoid biosynthesis in leaves, roots, and flower organs and responded towards drought and salt-stress conditions. Detailed analyses of PSY and 9-cis-epoxycarotenoid cleavage dioxygenase (MeNCED) expression and resulting ABA levels revealed MePSY1 as the main stress-responsive paralog. In the presence of high carotenoid levels in leaves, MePSY1 appeared to support, but not to be rate-limiting for ABA formation; MeNCED represented the main driver. The inverse situation was found in roots where carotenoid levels are low. Moreover, ABA formation and the relative induction kinetics showed discrimination between drought and salt stress. Compared to rice as a drought-intolerant species, the drought response in cassava followed a different kinetic regime. The difference is thought to represent a component contributing to the large differences in the adaptation towards water supply.

Insights from the cold transcriptome of Physcomitrella patens: global specialization pattern of conserved transcriptional regulators and identification of orphan genes involved in cold acclimation

The whole-genome transcriptomic cold stress response of the moss Physcomitrella patens was analyzed and correlated with phenotypic and metabolic changes. Based on time-series microarray experiments and quantitative real-time polymerase chain reaction, we characterized the transcriptomic changes related to early stress signaling and the initiation of cold acclimation. Transcription-associated protein (TAP)-encoding genes of P. patens and Arabidopsis thaliana were classified using generalized linear models. Physiological responses were monitored with pulse-amplitude-modulated fluorometry, high-performance liquid chromatography and targeted high-performance mass spectrometry. The transcript levels of 3220 genes were significantly affected by cold. Comparative classification revealed a global specialization of TAP families, a transcript accumulation of transcriptional regulators of the stimulus/stress response and a transcript decline of developmental regulators. Although transcripts of the intermediate to later response are from evolutionarily conserved genes, the early response is dominated by species-specific genes. These orphan genes may encode as yet unknown acclimation processes.

Tissue-Specific Apocarotenoid Glycosylation Contributes to Carotenoid Homeostasis in Arabidopsis Leaves

Apocarotenoid glycosides contribute to carotenoid biosynthetic pathway flux compensation. Attaining defined steady-state carotenoid levels requires balancing of the rates governing their synthesis and metabolism. Phytoene formation mediated by phytoene synthase (PSY) is rate limiting in the biosynthesis of carotenoids, whereas carotenoid catabolism involves a multitude of nonenzymatic and enzymatic processes. We investigated carotenoid and apocarotenoid formation in Arabidopsis (Arabidopsis thaliana) in response to enhanced pathway flux upon PSY overexpression. This resulted in a dramatic accumulation of mainly β-carotene in roots and nongreen calli, whereas carotenoids remained unchanged in leaves. We show that, in chloroplasts, surplus PSY was partially soluble, localized in the stroma and, therefore, inactive, whereas the membrane-bound portion mediated a doubling of phytoene synthesis rates. Increased pathway flux was not compensated by enhanced generation of long-chain apocarotenals but resulted in higher levels of C13 apocarotenoid glycosides (AGs). Using mutant lines deficient in carotenoid cleavage dioxygenases (CCDs), we identified CCD4 as being mainly responsible for the majority of AGs formed. Moreover, changed AG patterns in the carotene hydroxylase mutants lutein deficient1 (lut1) and lut5 exhibiting altered leaf carotenoids allowed us to define specific xanthophyll species as precursors for the apocarotenoid aglycons detected. In contrast to leaves, carotenoid hyperaccumulating roots contained higher levels of β-carotene-derived apocarotenals, whereas AGs were absent. These contrasting responses are associated with tissue-specific capacities to synthesize xanthophylls, which thus determine the modes of carotenoid accumulation and apocarotenoid formation.

Altered Dark- and Photoconversion of Phytochrome B Mediate Extreme Light Sensitivity and Loss of Photoreversibility of the phyB-401 Mutant

The phyB-401 mutant is 103 fold more sensitive to red light than its wild-type analogue and shows loss of photoreversibility of hypocotyl growth inhibition. The phyB-401 photoreceptor displays normal spectral properties and shows almost no dark reversion when expressed in yeast cells. To gain insight into the molecular mechanism underlying this complex phenotype, we generated transgenic lines expressing the mutant and wild-type phyB in phyB-9 background. Analysis of these transgenic lines demonstrated that the mutant photoreceptor displays a reduced rate of dark-reversion but normal Pfr to Pr photoconversion in vivo and shows an altered pattern of association/dissociation with nuclear bodies compared to wild-type phyB. In addition we show (i) an enhanced responsiveness to far-red light for hypocotyl growth inhibition and CAB2 expression and (ii) that far-red light mediated photoreversibility of red light induced responses, including inhibition of hypocotyl growth, formation of nuclear bodies and induction of CAB2 expression is reduced in these transgenic lines. We hypothesize that the incomplete photoreversibility of signalling is due to the fact that far-red light induced photoconversion of the chromophore is at least partially uncoupled from the Pfr to Pr conformation change of the protein. It follows that the phyB-401 photoreceptor retains a Pfr-like structure (Pr *) for a few hours after the far-red light treatment. The greatly reduced rate of dark reversion and the formation of a biologically active Pr * conformer satisfactorily explain the complex phenotype of the phyB-401 mutant and suggest that amino acid residues surrounding the position 564 G play an important role in fine-tuning phyB signalling.

Carotenogenesis Is Regulated by 5′UTR-Mediated Translation of Phytoene Synthase Splice Variants

A splice variant of Arabidopsis phytoene synthase is subjected to a feedback-mediated, translational control via its 5′UTR to coordinate biosynthetic pathway flux with carotenoid requirements. Phytoene synthase (PSY) catalyzes the highly regulated, frequently rate-limiting synthesis of the first biosynthetically formed carotene. While PSY constitutes a small gene family in most plant taxa, the Brassicaceae, including Arabidopsis (Arabidopsis thaliana), predominantly possess a single PSY gene. This monogenic situation is compensated by the differential expression of two alternative splice variants (ASV), which differ in length and in the exon/intron retention of their 5′UTRs. ASV1 contains a long 5′UTR (untranslated region) and is involved in developmentally regulated carotenoid formation, such as during deetiolation. ASV2 contains a short 5′UTR and is preferentially induced when an immediate increase in the carotenoid pathway flux is required, such as under salt stress or upon sudden light intensity changes. We show that the long 5′UTR of ASV1 is capable of attenuating the translational activity in response to high carotenoid pathway fluxes. This function resides in a defined 5′UTR stretch with two predicted interconvertible RNA conformations, as known from riboswitches, which might act as a flux sensor. The translation-inhibitory structure is absent from the short 5′UTR of ASV2 allowing to bypass translational inhibition under conditions requiring rapidly increased pathway fluxes. The mechanism is not found in the rice (Oryza sativa) PSY1 5′UTR, consistent with the prevalence of transcriptional control mechanisms in taxa with multiple PSY genes. The translational control mechanism identified is interpreted in terms of flux adjustments needed in response to retrograde signals stemming from intermediates of the plastid-localized carotenoid biosynthesis pathway.

Insights into the formation of carlactone from in‐depth analysis of the CCD8‐catalyzed reactions

Strigolactones are a new class of phytohormones synthesized from carotenoids via carlactone. The complex structure of carlactone is not easily deducible from its precursor, a cis‐configured β‐carotene cleavage product, and is thus formed via a poorly understood series of reactions and molecular rearrangements, all catalyzed by only one enzyme, the carotenoid cleavage dioxygenase 8 (CCD8). Moreover, the reactions leading to carlactone are expected to form a second, yet unidentified product. In this study, we used 13C and 18O‐labeling to shed light on the reactions catalyzed by CCD8. The characterization of the resulting carlactone by LC‐MS and NMR, and the identification of the assumed, less accessible second product allowed us to formulate a minimal reaction mechanism for carlactone generation.